Supplementary Material

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1 Supplementary Material The Cerato-Platanin protein Epl-1 from Trichoderma harzianum is involved in mycoparasitism, plant resistance induction and self cell wall protection Eriston Vieira Gomes 1, Mariana do Nascimento Costa 1, Renato Graciano de Paula 1, Rafael Ricci de Azevedo, Francilene Lopes da Silva, Eliane F. Noronha, Cirano José Ulhoa, Valdirene Neves Monteiro 5, Rosa Elena Cardoza 6, Santiago Gutiérrez 6, Roberto Nascimento Silva 1* 1 - Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil. - Department of Molecular and Cellular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil. - Department of Cellular Biology, University of Brasilia, Brasília, Distrito Federal, Brazil. - Department of Biochemistry and Cellular Biology, Biological Sciences Institute, Federal University of Goias, Goiânia, Goiás, Brazil. 5 - Department of Biochemistry, State University of Goias, Anápolis, Goiás, Brazil. 6 - Department of Microbiology, University School of Agricultural Engineers, University of León, Ponferrada, Spain. *Correspondence: Dr. Roberto do Nascimento Silva University of São Paulo Ribeirão Preto Medical School Department of Biochemistry and Immunology 9, Bandeirantes Av Ribeirão Preto, SP rsilva@fmrp.usp.br

2 1 - Supplementary Figures: Supplementary Figure 1: Analysis of potential O-glycosylation sites in T. harzianum Epl-1 protein sequence. The red horizontal line indicates the threshold potential O- glycosylation. The blue vertical lines indicate the position of the site in the protein sequence. Sites with blue vertical lines which cross the red threshold line have potential O-glycosylation. Supplementary Figure : Analysis of potential O-β-N-Acetil-Glicosilation sites in T. harzianum Epl-1 protein sequence. The horizontal blue line indicates the threshold potential O-β-N-Acetyl glycosylation. The green vertical lines indicate the position of the site in the protein sequence. Sites with green vertical lines exceeding the threshold blue line have a potential of O-β-N-Acetyl glycosylation.

3 Supplementary Figure : Analysis of potential phosphorylation sites on T. harzianum Epl-1 protein sequence. The horizontal gray line indicates the threshold phosphorylation potential. The vertical colored lines indicate the potential amino acid; the position of the potential amino acid in the protein sequence. Sites with vertical lines cross the threshold line present potential phosphorylation.

4 5 AT G epl- Supplementary Figure : Representation of predicted regulatory motifs in the promoter region of T. harzianum epl-1 gene. The numbers indicate the position relative to the ATG translation start codon. Arrows indicate the orientation of the motif in the sense (5 ' ') and antisense strands (5 ' ') respectively. CAAT box and TATAA box transcription initiation sites; MYC-1 Mycoparasitism Response Element -1; CreA Carbon response regulator; GCCARG ph regulatory protein site; CCCCT Stress response elements; HGATAR Global nitrogen regulation.

5 A B C Supplementary Figure 5: Transformants screening scheme. A Schematic representation of the genomic region containing the epl-1 gene and the respective annealing sites of mutant screening primers set (MSEpl-1) (arrows). B Mutant screening: agarose gel electrophoresis of epl-1 gene PCR amplification. WT - T. harzianum wild type (19 bp); 1 1- Screening of mitotically stable epl-1 transformants (57bp). C - Mutant screening agarose gel electrophoresis of hph gene PCR amplification. WT - T. harzianum wild type (no amplification); 1 1- Screening

6 of mitotically stable epl-1 transformants (6bp). Individuals marked with orange rectangle were selected for further analysis; (1kb) Molecular weight marker. Supplementary Figure 6: Southern Blot analysis. (A) Representation of epl-1 encoding gene in T. harzianum Wild Type strain with EcoRV restriction sites, the fragments formed after digestion with its respective size (vertical lines in black) and MSEpl-1 primers annealing sites for probe construction and hybridization; (B) Representation of epl-1 deletion cassette with EcoRV restriction sites. The fragments formed after digestion with their respective sizes (vertical black lines) and MSEpl-1 primers annealing sites for probe hybridization; (C) Southern Blot Analysis of total DNA from T. harzianum Wild Type (T.h WT) and T. harzianum Epl-1 deleted mutant (T. h - Epl-1) digested with EcoRV endonuclease; (ƛHindIII) Size Markers.

7 Supplementary Figure 7: T. harzianum RecEpl-1-GFP strain construction. A - Schematic representation of the Epl-1 recover cassette fused with GFP construction. B - T. harzianum RecEpl-1-GFP strain fluorescence microscopy, x magnification. C agarose gel electrophoresis of epl-1 gene PCR amplification. WT - T. harzianum wild type strain (66 bp); Th Epl-1 T. harzianum epl-1 strain (no amplification product); Th RecEpl-1-GFP T. harzianum epl-1 complemented strain (66 bp). Size Marker 1kb Molecular weight marker. D - Relative Expression levels (linear) of epl-1 gene in different T. harzianum strains. (Th) T. harzianum (WT); ( epl1) T. harzianum epl1; (RecEpl-1) T. harzianum RecEpl-1-GFP; (ND) No detected.

8 DAYS BC DAYS C 7 DAYS AC Supplementary Figure 8: Direct Confrontation Assay. (T) T. harzianum wild type; ( Epl-1) T. harzianum epl-1; (S) S. sclerotiorum; BC Before hyphae contact; C hyphae contact; AC After hyphae contact.

9 epl-1 1,-β-exoglucanase (exg1) A B C ND ND ND BC AC BC AC BC AC BC BC AC TxT TxS TxΔEpl1 ΔEpl1 SxΔEpl1 * 5 1 * ND ** BC AC BC AC BC AC BC BC AC TxT TxS TxΔEpl1 ΔEpl1 SxΔEpl1 1 α-mannosidase (gh9) ** D α-1,-glucosidase (mutaw) E acid phosphatase F * ** ** 9 6 ND 1 Phytase (phyat) Supplementary Figure 9: Relative Expression levels (linear) of T. harzianum mycoparasitism-related genes in direct confrontation assay. (T) T. harzianum (WT); (S) S. sclerotiorum; ( Epl1) T. harzianum epl1; (BC) before hyphae contact; (AC) after hyphae contact; (ND) No detected. The data were presented using the -ΔΔCt method. * p<.5, ** p<.1, p<.1.

10 Chitinase (nag1) Chitinase (chit) A B 6 C ** * ** 1 Chitinase (chit) D E F 6 5 Aspartyl Protease (papa) Trypsin-like Protease (PRA1) 5 Serine Protease (sprt) 6 * Supplementary Figure 1: Relative Expression of T. harzianum mycoparasitism-related genes in direct confrontation assay. (T) T. harzianum (WT); (S) S. sclerotiorum; ( Epl1) T. harzianum epl1; (NC) no hyphae contact; (C) with hyphae contact; (ND) No detected. The data were presented using the -ΔΔCt method.* p<.5, ** p<.1, p<.1.

11 A B A B Supplementary Figure 11: Construction of Epl-1 deletion vector. A - pbluescript SK+ vector with selection hph-cassette (pbshph) with its respective restriction sites. B Complete pbshphepl-1 deletion vector with promoter and terminator epl-1 region in its respective cloning sites.

12 Supplementary Figure 1: Schematic Bell et al., 198 modified method, to classify Trichoderma strains in antagonistic activity assay in plate. TRIC. T. harzianum strains. PAT. Pathogen strains. - Supplementary Videos Legends: Supplementary Video 1: Fluorescence microscopy of Trichoderma harzianum RecEpl-1-GFP strain hyphae in x optical magnification. Supplementary Video : Fluorescence microscopy of Trichoderma harzianum RecEpl-1-GFP strain hyphae in x optical magnification.